Multi-target drug candidates for the treatment of triple-negative breast cancer

ABSTRACT

Multitarget-directed compounds combining the features of reactive oxygen species (ROS) modulators, poly (ADP-ribose) polymerase 1 (PARP1), and/or heat shock protein 90 (Hsp90) inhibitors in a single molecule. These single agents constitute a systemic regimen for treating the triple-negative breast cancer phenotype that overcomes drug resistance and tumor recurrence, and may be used for other indications as well, such as the treatment of various neurodegenerative disorders.

BACKGROUND 1. Field

The disclosure of the present patent application relates to novelmultifunctional compounds combining the features of ROS modulators,PARP1, and/or Hsp90 inhibitors in a single molecule, in particular theuse of such compounds for treating triple-negative breast cancer.

2. Description of the Related Art

Triple-negative breast cancer (TNBC) accounts for 20-25% of all breastcancers and is characterized by its aggressive clinical behavior, poorprognosis, and high recurrence rates. TNBC lacks the overexpression ofthe estrogen receptor (ER), the progesterone receptor (PR), and thehuman epidermal growth factor 2 receptor (HER2), and therefore,cytotoxic chemotherapy remains the only therapeutic option. Althoughchemotherapy has shown to be effective in early-stage TNBC disease,patients in advanced disease states respond poorly to these drugs.Furthermore, drug resistance and a high risk of relapse were constantlyobserved in early and advanced stages. These are attributed to thecomplicated and multifaceted nature of TNBC, which results from combinedbiochemical alterations, sequential mutations, and modification in geneexpression occurring at various genetic levels and pathways. To targetthis multifactorial mechanistic nature of TNBC, novel multitarget drugcandidates are needed to interact with these multiple altered events andpathways.

Among the various TNBC alterations, BRCA1 dysregulation and increasedgeneration of reactive oxygen species (ROS) have recently emerged aspotential therapeutic targets. Further, recent genomic, biological, andmolecular investigations have shown that intrinsic TNBC heterogeneity isthe result of a combination of both genetic and biochemical alterations.These include low levels of hormone receptor-related genes (ER and PR),absence of HER2, BRCA1 dysregulation, and overexpression of specificproteins (e.g., Hsp90, EGFR, VEGF, p53, p15, and cyclin E in addition toglycolysis- and mitochondrial metabolism-related proteins). These areultimately associated with metabolic phenotype and mitochondrialdysfunction in addition to a disturbance in the intracellular redoxnetwork. The latter encompasses high levels of ROS, reactive nitrogenspecies (RNS), and metal ions as well as reduced antioxidant capacity.Therefore, the development of more effective TNBC therapies with highclinical impact requires an extensive understanding of the abovesophisticated biochemical and genetic abnormalities.

Heat shock protein 90 (Hsp90) inhibition has the potential tosimultaneously stop the function of proteins associated with highexpression TNBC recurrence and chemotherapy resistance and subsequentlysuppress tumor growth. In addition, Hsp90 inhibitors can overcome drugresistance due to secondary mutations of a target protein. On the otherhand, increased generation of ROS and oxidative stress (OS) were alsocommon features in TNBC. In this regard, ROS modulation can be exploitedtherapeutically for the preferential elimination of cancer cells.Although these targeted strategies have individually shown enhancedpreclinical treatment response, none of them were approved by the U.S.Food and Drug Administration (FDA). Moreover, no apparent, proven, andeffective single agent has been demonstrated to provide a systemicregimen recommended for treating the triple-negative phenotype thatovercomes drug resistance and tumor recurrence. Given the high morbidityand mortality of TNBC, the lack of available specific drugs necessitatesdeveloping more targeted and less toxic therapies to overcome drugresistance and expand the available effective drug arsenal to battlethis disease.

Thus, new multitarget drug candidates solving the aforementionedproblems are desired.

SUMMARY

The present subject matter relates to novel multifunctional compoundsthat can simultaneously interfere with multiple altered pathways, makingthem effective, for example, in the treatment of triple-negative breastcancer (TNBC). Consequently, the present subject matter relates tomultitarget-directed compounds combining the features of reactive oxygenspecies (ROS) modulators, poly (ADP-ribose) polymerase 1 (PARP1), and/orheat shock protein 90 (Hsp90) inhibitors in a single molecule.Accordingly, for the first time, the present subject matter relates toan effective single agent constituting a systemic regimen for treatingthe triple-negative breast cancer phenotype that overcomes drugresistance and tumor recurrence.

In an embodiment, the present subject matter relates to amulti-functional agent

having the formula I:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.

In another embodiment, the present subject matter relates to a method ofmaking the multi-functional agent of formula I, the method comprising:reacting, at room temperature, a quinazolin-2-ylmethanamine withbenzaldehyde in methanol, then adding a3-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid and a2-isocyanoethylselane to obtain the multifunctional agent according tothe following reaction scheme:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.

In another embodiment, the present subject matter relates to amulti-functional agent having the formula II:

wherein:R₄ is

R′ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an aryl group, aheteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈ cycloalkenylgroup; and Y is guanine, cytosine, or adenine.

In a further embodiment, the present subject matter relates to a methodof making the multi-functional agent of formula II, the methodcomprising: reacting 2-(phenylselanyl)acetaldehyde and tert-butylisocyanide with guanidine, cytosine, or adenine in DMSO according to thefollowing reaction scheme:

In an embodiment, the present subject matter relates to a pharmaceuticalcomposition, comprising a multi-functional agent as described herein anda pharmaceutically acceptable carrier.

In a further embodiment, the present subject matter relates to a methodof treating cancer in a patient, the method comprising: administering amulti-functional agent as described herein to a patient in need thereof.Similarly, the present subject matter also relates to a method oftreating a neurodegenerative disorder in a patient, the methodcomprising: administering a multi-functional agent as described hereinto a patient in need thereof.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a chart showing how TNBC cells are characterized byincreased production of ROS, decreased antioxidants, low expression ofhormone receptor (ER and PR) and HER-2-related genes, metabolicabnormalities and BRCA1 mutations.

FIG. 2 depicts a chart showing a multitarget-directed drug strategy as abasis for therapeutic selectivity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions are provided for the purpose of understandingthe present subject matter and for construing the appended patentclaims.

Definitions

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise. As used herein, the term “about” refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

As used herein, “alkyl” refers to a straight-chain or branched saturatedhydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl(Et), propyl (e.g., n-propyl and z′-propyl), butyl (e.g., n-butyl,z′-butyl, sec-butyl, tert-butyl), pentyl groups (e.g., n-pentyl,z′-pentyl, -pentyl), hexyl groups, and the like. In various embodiments,an alkyl group can have 1 to 40 carbon atoms (i.e., C₁—C₄₀ alkyl group),for example, 1-30 carbon atoms (i.e., C₁—C₃₀ alkyl group). In someembodiments, an alkyl group can have 1 to 6 carbon atoms, and can bereferred to as a “lower alkyl group” or a “C₁—C₆ alkyl group”. Examplesof lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl andz′-propyl), and butyl groups (e.g., n-butyl, z′-butyl, sec-butyl,tert-butyl). In some embodiments, alkyl groups can be substituted asdescribed herein. In other embodiments, alkyl groups can be optionallysubstituted as described herein. An alkyl group is generally notsubstituted with another alkyl group, an alkenyl group, or an alkynylgroup.

As used herein, “alkenyl” refers to a straight-chain or branched alkylgroup having one or more carbon-carbon double bonds. Examples of alkenylgroups include ethenyl, propenyl, butenyl, pentenyl, hexenyl,butadienyl, pentadienyl, hexadienyl groups, and the like. The one ormore carbon-carbon double bonds can be internal (such as in 2-butene) orterminal (such as in 1-butene). In various embodiments, an alkenyl groupcan have 2 to 40 carbon atoms (i.e., C₂—C₄₀ alkenyl group), for example,2 to 20 carbon atoms (i.e., C₂—C₂₀ alkenyl group) or 2 to 6 carbon atoms(i.e., C₂—C₆ alkenyl group). In some embodiments, alkenyl groups can besubstituted as described herein. In other embodiments, alkenyl groupscan be optionally substituted as described herein. An alkenyl group isgenerally not substituted with another alkenyl group, an alkyl group, oran alkynyl group.

The term “substituted alkyl” as used herein refers to an alkyl group inwhich 1 or more (up to about 5, for example about 3) hydrogen atoms isreplaced by a substituent independently selected from the group: —O, —S,acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino (wherein the amino group may be a cyclic amine), azido, carboxyl,(optionally substituted alkoxy)carbonyl, amido, cyano, optionallysubstituted cycloalkyl, optionally substituted cycloalkenyl, halogen,hydroxyl, nitro, sulfamoyl, sulfanyl, sulfinyl, sulfonyl, and sulfonicacid. Some of the optional substituents for alkyl are hydroxy, halogenexemplified by chloro and bromo, acyl exemplified by methylcarbonyl;alkoxy, and heterocyclyl exemplified by morpholino and piperidino. Otheralkyl substituents as described herein may further be contemplated. Theterm “substituted alkenyl” as used herein refers to an alkenyl group inwhich 1 or more (up to about 5, for example about 3) hydrogen atoms isreplaced by a substituent independently selected from those listed abovewith respect to a substituted alkyl. Other alkenyl substituents asdescribed herein may further be contemplated.

As used herein, “heteroatom” refers to an atom of any element other thancarbon or hydrogen and includes, for example, nitrogen, oxygen, silicon,sulfur, phosphorus, and selenium.

As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ringsystem or a polycyclic ring system in which two or more aromatichydrocarbon rings are fused (i.e., having a bond in common with)together or at least one aromatic monocyclic hydrocarbon ring is fusedto one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl groupcan have 6 to 24 carbon atoms in its ring system (e.g., C6-24 arylgroup), which can include multiple fused rings. In some embodiments, apolycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ringposition of the aryl group can be covalently linked to the definedchemical structure. Examples of aryl groups having only aromaticcarbocyclic ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl(bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic),pentacenyl (pentacyclic), and like groups. Examples of polycyclic ringsystems in which at least one aromatic carbocyclic ring is fused to oneor more cycloalkyl and/or cycloheteroalkyl rings include, among others,benzo derivatives of cyclopentane (i.e., an indanyl group, which is a5,6-bicyclic cycloalkyl/aromatic ring system), cyclohexane (i.e., atetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl/aromaticring system), imidazoline (i.e., a benzimidazolinyl group, which is a5,6-bicyclic cycloheteroalkyl/aromatic ring system), and pyran (i.e., achromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic ringsystem). Other examples of aryl groups include benzodioxanyl,benzodioxolyl, chromanyl, indolinyl groups, and the like. In someembodiments, aryl groups can be substituted as described herein. In someembodiments, an aryl group can have one or more halogen substituents,and can be referred to as a “haloaryl” group. Perhaloaryl groups, i.e.,aryl groups where all of the hydrogen atoms are replaced with halogenatoms (e.g., —C₆F₅), are included within the definition of “haloaryl”.In certain embodiments, an aryl group is substituted with another arylgroup and can be referred to as a biaryl group. Each of the aryl groupsin the biaryl group can be substituted, or optionally substituted, asdisclosed herein.

As used herein, “heteroaryl” refers to an aromatic monocyclic ringsystem containing at least one ring heteroatom selected from oxygen (O),nitrogen (N), sulfur (S), silicon (Si), and selenium (Se) or apolycyclic ring system where at least one of the rings present in thering system is aromatic and contains at least one ring heteroatom.Polycyclic heteroaryl groups include those having two or more heteroarylrings fused together, as well as those having at least one monocyclicheteroaryl ring fused to one or more aromatic carbocyclic rings,non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkylrings. A heteroaryl group, as a whole, can have, for example, 5 to 24ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 memberedheteroaryl group). The heteroaryl group can be attached to the definedchemical structure at any heteroatom or carbon atom that results in astable structure. Generally, heteroaryl rings do not contain O—O, S—S,or S—O bonds. However, one or more N or S atoms in a heteroaryl groupcan be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiopheneS,S-dioxide). Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl),SiH₂, SiH(alkyl), Si(alkyl)₂, SiH(arylalkyl), Si(arylalkyl)₂, orSi(alkyl)(arylalkyl). Examples of such heteroaryl rings includepyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl,thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl,benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl,quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl,naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl,pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl,thienoxazolyl, thienoimidazolyl groups, and the like. Further examplesof heteroaryl groups include 4,5,6,7-tetrahydroindolyl,tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups,and the like. In some embodiments, heteroaryl groups can be substituted,or optionally substituted, as described herein.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined herein.

It will be understood by those skilled in the art with respect to anychemical group containing one or more substituents that such groups arenot intended to introduce any substitution or substitution patterns thatare sterically impractical and/or physically non-feasible.

The term “isomers” or “stereoisomers” as used herein relates tocompounds that have identical molecular formulae but that differ in thearrangement of their atoms in space. Stereoisomers that are not mirrorimages of one another are termed “diastereoisomers” and stereoisomersthat are non-superimposable mirror images are termed “enantiomers,” orsometimes optical isomers. A carbon atom bonded to four non-identicalsubstituents is termed a “chiral center.” Certain compounds herein haveone or more chiral centers and therefore may exist as either individualstereoisomers or as a mixture of stereoisomers. Configurations ofstereoisomers that owe their existence to hindered rotation about doublebonds are differentiated by their prefixes cis and trans (or Z and E),which indicate that the groups are on the same side (cis or Z) or onopposite sides (trans or E) of the double bond in the molecule accordingto the Cahn-Ingold-Prelog rules. All possible stereoisomers arecontemplated herein as individual stereoisomers or as a mixture ofstereoisomers. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which the presently described subjectmatter pertains. Accordingly, “isomers” or “stereoisomers” of allcompounds described herein are contemplated as falling within the scopeof the presently disclosed subject matter.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

As described herein, “room temperature” means a temperature of about 25°C.

The present subject matter relates to novel multifunctional compoundsthat can simultaneously interfere with multiple altered pathways, makingthem effective, for example, in the treatment of triple-negative breastcancer (TNBC). Consequently, the present subject matter relates tomultitarget-directed compounds combining the features of reactive oxygenspecies (ROS) modulators, poly (ADP-ribose) polymerase 1 (PARP1), and/orheat shock protein 90 (Hsp90) inhibitors in a single molecule.Accordingly, for the first time, the present subject matter relates toan effective single agent constituting a systemic regimen for treatingthe triple-negative breast cancer phenotype that overcomes drugresistance and tumor recurrence.

Although PARP1 and Hsp90 inhibitors seem to have different therapeutictargets than ROS modulators, both run on the same track. Increased ROSgeneration by ROS modulators leads to DNA double-strand breaks, which isquite similar to the molecular mode of action displayed by PARP1inhibitors. At the same time, these ROS modulators have the potential tocleave and deactivate Hsp90. The value of synthetic libraries relies ontheir feasibility (easy access) and their probability to hit thebiological target(s), which is related to their diversity andcomplexity.

It has been a major challenge to install not just one or two but threeor more distinct pharmacophores in one single molecule. A one-potsingle-step synthetic strategy using stable reagents under neutralconditions is therefore employed herein, i.e., the isocyanide-basedmulticomponent (IMCR) Ugi and Groebke-Blackburn-Bienayme (GBB)reactions, as described in more detail below.

In an embodiment, the present subject matter relates to amulti-functional agent having the formula I:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.

In an embodiment, the present subject matter relates to amulti-functional agent of formula I, wherein: R₁ is a C₁—C₆ alkyl group;R₂ is H; and R₃ is an aryl group.

In another embodiment, the present subject matter relates to amulti-functional agent of formula I, having the formula:

In an embodiment, the present subject matter relates to amulti-functional agent having the formula II:

wherein:R₄ is

R′ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an aryl group, aheteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈ cycloalkenylgroup; and Y is guanine, cytosine, or adenine.

In a further embodiment, the present subject matter relates to amulti-functional agent of formula II, wherein R₄ is

In still another embodiment, the present subject matter relates to amulti-functional agent of formula II, selected from the group consistingof:

It is to be understood that the present subject matter covers allcombinations of substituent groups referred to herein.

The present compounds may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Accordingly, the present subjectmatter includes all solvates of the present compounds of formulae I andII and pharmaceutically acceptable stereoisomers, esters, and/or saltsthereof. Hydrates are one example of such solvates.

Further, the present subject matter includes all mixtures of possiblestereoisomers of the embodied compounds, independent of the ratio,including the racemates.

Salts of the present compounds, or the salts of the stereoisomersthereof, include all inorganic and organic acid addition salts and saltswith bases, especially all pharmaceutically acceptable inorganic andorganic acid addition salts and salts with bases, particularly allpharmaceutically acceptable inorganic and organic acid addition saltsand salts with bases customarily used in pharmacy.

Examples of acid addition salts include, but are not limited to,hydrochlorides, hydrobromides, phosphates, nitrates, sulfates, acetates,trifluoroacetates, citrates, D-gluconates, benzoates, 2-(4-hydroxy-benzoyl)benzoates, butyrates, subsalicylates, maleates, laurates,malates, lactates, fumarates, succinates, oxalates, tartrates,stearates, benzenesulfonates (besilates), toluenesulfonates (tosilates),methanesulfonates (mesilates) and 3-hydroxy-2-naphthoates.

Examples of salts with bases include, but are not limited to, lithium,sodium, potassium, calcium, aluminum, magnesium, titanium, ammonium,meglumine and guanidinium salts. The salts include water-insoluble and,particularly, water-soluble salts.

The present compounds, the salts, the stereoisomers and the salts of thestereoisomers thereof may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Included within the present scopeare, therefore, all solvates of the compounds of formulae I and II, aswell as the solvates of the salts, the stereoisomers and the salts ofthe stereoisomers of the compounds of formulae I and II.

In obtaining the present compounds in their solvate form, the presentcompounds may be isolated and purified in a manner known per se, e.g.,by distilling off the solvent in vacuo and recrystallizing the residueobtained from a suitable solvent or subjecting it to one of thecustomary purification methods, such as column chromatography on asuitable support material.

Salts of the compounds of formulae I and II and the stereoisomersthereof can be obtained by dissolving the free compound in a suitablesolvent (by way of non-limiting example, a ketone such as acetone,methylethylketone or methylisobutylketone; an ether such as diethylether, tetrahydrofurane or dioxane; a chlorinated hydrocarbon such asmethylene chloride or chloroform; a low molecular weight aliphaticalcohol such as methanol, ethanol or isopropanol; a low molecular weightaliphatic ester such as ethyl acetate or isopropyl acetate; or water)which contains the desired acid or base, or to which the desired acid orbase is then added. The acid or base can be employed in saltpreparation, depending on whether a mono- or polybasic acid or base isconcerned and depending on which salt is desired, in an equimolarquantitative ratio or one differing therefrom. The salts are obtained byfiltering, reprecipitating, precipitating with a non-solvent for thesalt or by evaporating the solvent. Salts obtained can be converted intothe free compounds which, in turn, can be converted into salts. In thismanner, pharmaceutically unacceptable salts, which can be obtained, forexample, as process products in the manufacturing on an industrialscale, can be converted into pharmaceutically acceptable salts byprocesses known to the person skilled in the art.

Pure diastereomers and pure enantiomers of the present compounds can beobtained, e.g., by asymmetric synthesis, by using chiral startingcompounds in synthesis and by splitting up enantiomeric anddiastereomeric mixtures obtained in synthesis. Preferably, the purediastereomeric and pure enantiomeric compounds are obtained by usingchiral starting compounds in synthesis.

Enantiomeric and diastereomeric mixtures can be split up into the pureenantiomers and pure diastereomers by methods known to a person skilledin the art. Preferably, diastereomeric mixtures are separated bycrystallization, in particular fractional crystallization, orchromatography. Enantiomeric mixtures can be separated, e.g., by formingdiastereomers with a chiral auxiliary agent, resolving the diastereomersobtained and removing the chiral auxiliary agent. As chiral auxiliaryagents, for example, chiral acids can be used to separate enantiomericbases and chiral bases can be used to separate enantiomeric acids viaformation of diastereomeric salts. Furthermore, diastereomericderivatives such as diastereomeric esters can be formed fromenantiomeric mixtures of alcohols or enantiomeric mixtures of acids,respectively, using chiral acids or chiral alcohols, respectively, aschiral auxiliary agents. Additionally, diastereomeric complexes ordiastereomeric clathrates may be used for separating enantiomericmixtures. Alternatively, enantiomeric mixtures can be split up usingchiral separating columns in chromatography. Another suitable method forthe isolation of enantiomers is enzymatic separation.

In one embodiment, the present subject matter relates to the use of aone-pot single-step synthetic method, i.e., the isocyanide-basedmulticomponent (IMCR) Ugi and Groebke-Blackburn-Bienayme (GBB)reactions, to prepare the present multi-functional agents. Thesereactions are promising synthetic tools in the context of DiversityOriented Synthesis and are used to achieve high levels of efficiency,complexity, and diversity in a single step (short reaction sequences:brevity) by varying each component participating in the reaction.Combining simple and flexible building blocks gives rise to the presentnovel complex structures by the simultaneous formation of two or morebonds.

Moreover, the present synthetic methods are notable for their ease ofautomation, simplicity, superior atom economy, ability to obtain a largenumber of compounds with minimal effort, reduced reaction time and cost,and formation of pure products with avoidance of tedious workup andpurification.

By using readily available quinone, selenium, quinazoline, and/orpurines and/or imidazopyridines building blocks in Ugi reactions,diverse libraries of structurally rather complex ROS modulators can besynthesized. One additional advantage of these specific types of I-MCRs,the product nature, i.e., typically carboxamides (a-acylaminocarboxamides), are obtained which are common structures in many PARP1inhibitors. So, in this case, these I-MCRs would deliver libraries ofROS modulators/Hsp90 with expected PARP1 inhibitor activity.

For example, diverse libraries of structurally rather complex PARP1inhibitors and ROS modulators can be synthesized using the readilyavailable selenoquinone (1) and amino quinazoline (2) in the Ugireaction. So, in this case, this strategy would deliver libraries of ROSmodulators and PARP1 inhibitor activity.

Accordingly, in an embodiment, the present subject matter relates to amethod of making the multi-functional agent of formula I, the methodcomprising: reacting, at room temperature, a quinazolin-2-ylmethanaminewith benzaldehyde in methanol, then adding a3-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid and a2-isocyanoethylselane to obtain the multifunctional agent according tothe following reaction scheme:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.

In another embodiment, the present subject matter relates to a method ofmaking a multi-functional agent having the formula:

the method comprising: reacting, at room temperature, aquinazolin-2-ylmethanamine with benzaldehyde in methanol, then adding a3-((3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acidand a (2-isocyanoethyl)(phenyl)selane to obtain the multifunctionalagent according to the following reaction scheme:

In certain embodiments in this regard, the quinazolin-2-ylmethanamine,benzaldehyde, 3 -((3 -methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid, and(2-isocyanoethyl)(phenyl)selane can be reacted in an about 1:1.1:1.1:1.1molar ratio.

In a further embodiment, the present subject matter relates to a methodof making the multi-functional agent of formula II, the methodcomprising: reacting 2-(phenylselanyl)acetaldehyde and tert-butylisocyanide with guanidine, cytosine, or adenine in DMSO according to thefollowing reaction scheme:

In this regard, in the method for making compound 9 shown above, thereaction can occur in DMSO under nitrogen at a temperature of about 65°C. to about 75° C., about 65° C., about 66° C., about 67° C., about 68°C., about 69° C., about 70° C., about 71° C., about 72° C., about 73°C., about 74° C., about 75° C., or about 70° C., for at least about 8hour.

In certain embodiments in this regard, the tert-butyl isocyanide andguanidine, cytosine, or adenine are reacted in an about 1:1.1 molarratio.

In an embodiment, the present subject matter relates to a pharmaceuticalcomposition, comprising a therapeutically effective amount of amulti-functional agent as described herein and a pharmaceuticallyacceptable carrier. In this regard, the pharmaceutically acceptablecarrier can be one or more pharmaceutically acceptable carriers,excipients, or vehicles. In some embodiments, the present compositionscan be used for combination therapy, where other therapeutic and/orprophylactic ingredients can be included therein.

In an embodiment, the pharmaceutical composition comprises one or two ofthe present multi-functional agents, or one of the presentmulti-functional agents.

Non-limiting examples of suitable excipients, carriers, or vehiclesuseful herein include liquids such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, and the like. Suitableexcipients for nonliquid formulations are also known to those of skillin the art. A thorough discussion of pharmaceutically acceptableexcipients and salts useful herein is available in Remington'sPharmaceutical Sciences, 23rd Edition. Easton, Pa., Mack PublishingCompany, 2020, the entire contents of which are incorporated byreference herein.

The present multi-functional agents are typically administered at atherapeutically or pharmaceutically effective dosage, e.g., a dosagesufficient to provide treatment for, e.g., a cancer or aneurodegenerative disorder. Administration of the multi-functionalagents or pharmaceutical compositions thereof can be by any method thatdelivers the multi-functional agents systemically and/or locally. Thesemethods include oral routes, parenteral routes, intraduodenal routes,and the like.

While human dosage levels have yet to be optimized for the presentmulti-functional agents, generally, a daily dose is from about 0.01 to10.0 mg/kg of body weight, for example about 0.1 to 5.0 mg/kg of bodyweight. The precise effective amount will vary from subject to subjectand will depend upon the species, age, the subject's size and health,the nature and extent of the condition being treated, recommendations ofthe treating physician, and the therapeutics or combination oftherapeutics selected for administration. The subject may beadministered as many doses as is required to reduce and/or alleviate thesigns, symptoms, or causes of the disease or disorder in question, orbring about any other desired alteration of a biological system.

In employing the present multi-functional agents for treatment of adisease, disorder, or condition, any pharmaceutically acceptable mode ofadministration can be used with other pharmaceutically acceptableexcipients, including solid, semi-solid, liquid or aerosol dosage forms,such as, for example, tablets, capsules, powders, liquids, suspensions,suppositories, aerosols or the like. The present multi-functional agentscan also be administered in sustained or controlled release dosageforms, including depot injections, osmotic pumps, pills, transdermal(including electrotransport) patches, and the like, for the prolongedadministration of the multi-functional agents at a predetermined rate,preferably in unit dosage forms suitable for single administration ofprecise dosages.

The present multi-functional agents may also be administered ascompositions prepared as foods for foods or animals, including medicalfoods, functional food, special nutrition foods and dietary supplements.A “medical food” is a product prescribed by a physician that is intendedfor the specific dietary management of a disorder or health conditionfor which distinctive nutritional requirements exist and may includeformulations fed through a feeding tube (referred to as enteraladministration or gavage administration).

A “dietary supplement” shall mean a product that is intended tosupplement the human diet and may be provided in the form of a pill,capsule, tablet, or like formulation. By way of non-limiting example, adietary supplement may include one or more of the following dietaryingredients: vitamins, minerals, herbs, botanicals, amino acids, anddietary substances intended to supplement the diet by increasing totaldietary intake, or a concentrate, metabolite, constituent, extract, orcombinations of these ingredients, not intended as a conventional foodor as the sole item of a meal or diet. Dietary supplements may also beincorporated into foodstuffs, such as functional foods designed topromote control of glucose levels. A “functional food” is an ordinaryfood that has one or more components or ingredients incorporated into itto give a specific medical or physiological benefit, other than a purelynutritional effect. “Special nutrition food” means ingredients designedfor a particular diet related to conditions or to support treatment ofnutritional deficiencies.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,for example about 0.5% to 50%, by weight of the present multi-functionalagents, the remainder being suitable pharmaceutical excipients,carriers, etc.

One manner of administration for the conditions detailed above is oral,using a convenient daily dosage regimen which can be adjusted accordingto the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like. Such compositions take theform of solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

The present compositions may take the form of a pill or tablet and thusthe composition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose and derivativesthereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active multi-functionalagent as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, or solubilizingagents, pH buffering agents and the like, for example, sodium acetate,sodium citrate, cyclodextrin derivatives, sorbitan monolaurate,triethanolamine acetate, triethanolamine oleate, etc.

For oral administration, a pharmaceutically acceptable non-toxiccomposition may be formed by the incorporation of any normally employedexcipients, such as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, talcum, cellulose derivatives,sodium croscarmellose, glucose, sucrose, magnesium carbonate, sodiumsaccharin, talcum and the like. Such compositions take the form ofsolutions, suspensions, tablets, capsules, powders, sustained releaseformulations and the like.

For a solid dosage form, a solution or suspension in, for example,propylene carbonate, vegetable oils or triglycerides, may beencapsulated in a gelatin capsule. Such diester solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545, the contents of each of which areincorporated herein by reference. For a liquid dosage form, thesolution, e.g., in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active multi-functional agent in vegetableoils, glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

Other useful formulations include those set forth in U.S. Pat. Nos. Re.28,819 and 4,358,603, the contents of each of which are herebyincorporated by reference.

Another manner of administration is parenteral administration, generallycharacterized by injection, either subcutaneously, intramuscularly orintravenously. Injectables can be prepared in conventional forms, eitheras liquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.

Another approach for parenteral administration employs the implantationof a slow-release or sustained-release system, such that a constantlevel of dosage is maintained. The percentage of active multi-functionalagent contained in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the activity of themulti-functional agent and the needs of the subject. However,percentages of active ingredient of 0.01% to 10% in solution areemployable and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. The composition maycomprise 0.2% to 2% of the active agent in solution.

Nasal solutions of the active multi-functional agent alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

Formulations of the active multi-functional agent may also beadministered to the respiratory tract as an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation have diameters of less than 50 microns, forexample less than 10 microns.

In a further embodiment, the present subject matter relates to a methodof treating cancer in a patient, the method comprising: administering amulti-functional agent as described herein to a patient in need thereof.In certain embodiments, the cancer can be triple-negative breast cancer(TNBC).

In this regard, TNBC has been a particularly difficult disease, and formof breast cancer, to treat as TNBC cells are characterized by increasedproduction of ROS, decreased antioxidants, low expression of hormonereceptor (ER and PR) and HER-2-related genes, metabolic abnormalitiesand BRCA1 mutations, as shown in FIG. 1 . Accordingly, the presentmulti-functional agents are uniquely suited to treat TNBC, as theseagents combine the features of ROS modulators, poly (ADP-ribose)polymerase 1 (PARP1), and/or heat shock protein 90 (Hsp90) inhibitors ina single molecule. In particular, TNBC should be particularlysusceptible to a multitarget drug strategy as a basis for therapeuticselectivity involving cell death-associated mechanisms of combinatorialROS modulators and PARP1 and Hsp90 inhibitors. Such methods of treatmentwere previously only available by the combined use of multiple activeagents, whereas the present multi-functional agents for the first timeprovide treatment involving all these pathways by administration of asingle active agent.

Although individual PARP1 and Hsp90 inhibitors have differenttherapeutic targets than ROS modulators, the combination strategyemployed herein can have a synergetic effect. Increased ROS generationby ROS modulators leads to DNA double-strand breaks, similar to themolecular mode of action displayed by PARP1 inhibitors. However, at thesame time, these ROS have the potential to cleave and deactivate Hsp90,as shown in FIG. 2 .

This strategy has several advantages over conventional methods. By wayof non-limiting example, the present multi-functional agents aredesigned to be compatible with the harsh TNBC microenvironmentconditions (e.g., OS, genetic alterations). Further, the use of thepresent multi-functional agents allows targets to be carefully chosen toselectively induce cancer cell death with minimal toxic effect to normalcells. In addition, blocking Hsp90 can lead to reduction of drugresistance and metastasis because the mutated/upregulated oncogenicproteins are usually Hsp90 clients. Likewise, combining two or morepharmacophores with a different therapeutic target(s) in one singlemolecule, as has been achieved by the present multi-functional agents,can synergistically potentiate their corresponding chemotherapeuticcytotoxicity, thereby allowing them to be introduced at much lowerconcentrations than usual. Finally, this strategy is not limited toTNBC, but it can also be applied to other cancer cells or diseasescomprising similar clinical behavior (e.g., neurodegenerativedisorders).

Accordingly, the present subject matter also relates to a method oftreating a neurodegenerative disorder in a patient, the methodcomprising: administering a multi-functional agent as described hereinto a patient in need thereof. In certain embodiments, theneurodegenerative disorder can be one or more of Parkinson's Disease,Alzheimer' s Disease, Huntington's Disease, Amyotrophic LateralSclerosis (ALS), and motor neuron disease. In addition, administrationof the present multi-functional agents to a patient can treat both thecancer and the neurodegenerative disorder simultaneously. The presentteachings are illustrated by the following examples.

EXAMPLES Example 1 PARP1 Inhibitors and ROS Modulators

The synthesis of ROS modulators can include quinones and organoseleniumscaffolds as described in Scheme 1. Additionally, two classes of PARP1inhibitors can be integrated in the synthesis strategy. These involvecarboxamide and quinazoline pharmacophores. These compounds can exhibitstructural similarities to the natural PARP-1 enzyme substrate,nicotinamide adenine dinucleotide (NAD⁺), and thus exert their inhibitoractivity by occupying the nicotinamide pocket.

The target compound 4 was synthesized according to the Ugiisocyanide-based multicomponent strategy. The reaction started with thereaction of quinazolin-2-ylmethanamine (1) (1 mmol) with benzaldehyde(1.1 mmol) in methanol (1 ml), followed by the addition of3-((3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid(2) (1.1 mmol) and (2-isocyanoethyl)(phenyl)selane (3) (1.1 mmol) atroom temperature.

Example 2 Hsp90 Inhibitors and ROS Modulators

Synthetic purines and imidazopyridines are used as small molecule Hsp90inhibitors as they mimic the purine base of the ADP/ATP andcompetitively bind to Hsp90, thereby blocking ATP binding to Hsp90 andinhibiting Hsp90 function. Therefore, GBBR was used for the synthesis ofaminoimidazole-condensed nucleobases (Hsp90 inhibitors) by the inclusionof the 2-amidine functionality of adenine, guanine or cytosine as shownin Scheme 2.

Scheme 2 represents the synthesis of purines and imidazopyridines whichin turn are Hsp90 inhibitors as they mimic the purine base of theADP/ATP and competitively bind to Hsp90, thereby blocking ATP binding toHsp90 and inhibiting Hsp90 function.

The target compounds 7, 8, and 9 were synthesized according to the GBBisocyanide-based multicomponent strategy. The reaction started by thereaction of 2-(phenylselanyl)acetaldehyde (5), tert-butyl isocyanide (6)(1 mmol) with either guanine, cytosine, or adenine (1.1 mmol) in DMSO (1ml) under nitrogen and at 70° C. for 8 hrs.

It is to be understood that the multi-functional agents are not limitedto the specific embodiments described above, but encompass any and allembodiments within the scope of the generic language of the followingclaims enabled by the embodiments described herein, or otherwise shownin the drawings or described above in terms sufficient to enable one ofordinary skill in the art to make and use the claimed subject matter.

We claim:
 1. A multi-functional agent having the formula I:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.
 2. The multi-functional agent of claim 1,wherein: R₁ is a C₁—C₆ alkyl group; R₂ is H; and R₃ is an aryl group. 3.The multi-functional agent of claim 1, having the formula:


4. A method of making the multi-functional agent of claim 1, the methodcomprising: reacting, at room temperature, a quinazolin-2-ylmethanaminewith benzaldehyde in methanol, then adding a3-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid and a2-isocyanoethylselane to obtain the multifunctional agent according tothe following reaction scheme:

wherein: R₁ is a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group, an arylgroup, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; R₂ is H, a C₁—C₆ alkyl group, a C₂—C₆ alkenyl group,an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or a C₃—C₈cycloalkenyl group; and R₃ is a C₁—C₆ alkyl group, a C₂—C₆ alkenylgroup, an aryl group, a heteroaryl group, a C₃—C₈ cycloalkyl group, or aC₃—C₈ cycloalkenyl group.
 5. A method of making the multi-functionalagent of claim 3, the method comprising: reacting, at room temperature,a quinazolin-2-ylmethanamine with benzaldehyde in methanol, then addinga 3-((3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acidand a (2-isocyanoethyl)(phenyl)selane to obtain the multifunctionalagent according to the following reaction scheme:


6. The method of claim 5, wherein the quinazolin-2-ylmethanamine,benzaldehyde,3-((3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)propanoic acid,and (2-isocyanoethyl) (phenyl)selane are reacted in an about1:1.1:1.1:1.1 molar ratio.
 7. A pharmaceutical composition, comprising atherapeutically effective amount of the multi-functional agent of claim1 and a pharmaceutically acceptable carrier.